Animals use the sense of taste to make decisions regarding potential food; substances with high nutritional value are ingested, while toxins and harmful substances are rejected. Interestingly, these behaviors are common across many species. Flies respond to sweet and bitter tastants with different stereotyped behaviors: sweet substances, often calorie rich, are appetitive and accepted, while bitter compounds, usually harmful, are rejected and avoided. The linkage between stimulus quality and behavioral response suggests that sweet and bitter tastants are represented differently in the brain. Mice process information regarding sweet and bitter substances in parallel through labeled lines. By contrast, in moths, a distributed combinatorial code for individual tastants was described, suggesting that the neural circuits are convergent. It is currently unknown which of these distinct models is operative in flies. Addressing this question will require a comprehensive analysis of the gustatory circuits layer by layer. While our understanding of the first-order level within the bitter and sweet circuits is rather advanced, little is known about neurons in the second-order level of the gustatory system. Most of the second-order neurons that have been characterized thus far have been identified by genetic screens. Due to the distributive nature of the first-order gustatory projections, one cannot identify the second-order neurons by the location of their dendrites, as has been done successfully in the olfactory circuits. In addition, flies have gustatory neurons in various parts of their body, and we hypothesize that a somatotopic gustatory map exists in the brain. All of these important gaps of knowledge would benefit from a robust genetic system for transsynaptic labeling of neural circuits. We have recently developed a new method for transsynaptic tracing and manipulation of neural circuits termed trans-Tango. We have validated trans-Tango in the olfactory system of flies and established it in the gustatory circuits that process information regarding sweet compounds. Our analysis revealed that second- order neurons in the sweet circuits project to neuromodulatory areas in the brain, some of which are known to be involved in controlling feeding behavior. Here we propose to implement trans-Tango to identify second- order projections in the bitter circuits. Our preliminary data suggest that the second-order projections in the bitter circuits are very similar to the second-order sweet projections. We propose a multipronged strategy that involves anatomical, functional and behavioral analyses aimed at characterizing in detail the second- and third- order projections within the sweet and bitter circuits. For our analysis, we will establish new versions of trans- Tango that incorporate new modules for functional analysis of circuits via calcium imaging and optogenetics, for intersectional connectivity studies, and for multicolor projection analysis. Thus, our studies will deepen our understanding of gustatory information processing in flies, a topic of high importance for human health in view of the relevance of the sense of taste for the role of insects as major vectors of many insect-born diseases.